Experimental
Hematology
Exp. Hematol. 15:1-9 (1987)
© 1987 International Society for Experimental Hematology
Hemopoietic Responses in Mice Injected with Purified Recombinant
Murine GM-CSF
Donald Metcalf, 1 C. Glenn Begley, 1 D. James Williamson, 1 Edouard C. Nice, 2 John De Lamarter, 3 JeanJacque Mermod, 3 David Thatcher, 3 and Albert Schmidt 3
1Cancer Research Unit, Walter and Eliza Hall Institute ofMedical Research, and 2 Ludwig Institute for Cancer Research, Royal Melbourne
Hospital, Melbourne, Victoria, Australia; and 3 Biogen SA, Geneva, Switzerland
(Received 13 May 1986; in revised form 10 July 1986; accepted 17 July 1986)
Abstract. Normal adult BALB/c, C57BL, and
C3H/HeJ mice were injected intraperitoneally three
times daily for six days with 6-200 ng purified, bacterially synthesized, murine recombinant GM-CSF.
Mice injected with 200 ng rGM-CSF developed a
twofold increase in blood neutrophils. In the peritoneal cavity, a dose-related rise was observed in
macrophages (up to 15-fold), neutrophils (10- to
100-fold) and eosinophils (10- to 100-fold). Peritoneal macrophages exhibited 15-fold increased mitotic activity (to 7.6/10 3 cells) and increased phagocytic activity for antibody-coated erythrocytes.
Increased numbers of infiltrating neutrophils and
monocytes were observed in the liver and lung. Doserelated rises were observed in spleen weight (up to
50%) and the spleen content ofmonocytes (twofold)
and nonerythroid progenitor cells (up to fourfold).
A dose-related fall occurred in total marrow cellularity (40%) and total nonerythroid progenitor cells
(37%-66%), but levels ofneutrophils and monocytes
remained constant. The data indicate that the injection ofrGM-CSF to normal mice increases overall numbers of granulocytes and macrophages and
the phagocytic activity of macrophages and provides direct evidence for the conclusion that GMCSF is likely to function in vivo as a regulator of
these cell populations.
Key words: Colony-stimulating factor - In vivo effects
Granulocyte-macrophage production - Phagocytosis
The colony-stimulating factors (CSFs) are a group
of specific glycoproteins that, in cultures of hemopoietic cells, are able to control the proliferation and
differentiation of granulocytes, macrophages, and
some related hemopoietic cell populations. Four
murine CSFs have been identified-GM-CSF
G-CSF, M-CSF (CSF-1), and Multi-CSF (IL-3)_:
----Address reprint requests to: Dr. D. Metcalf, Cancer Research Unit,
.Walt"r and Eliza Hall Institute of Medical Research, PO 3050,
Royal i\1elbourne Hospital, Victoria, Australia.
and each has been purified to homogeneity and its
range of actions in vitro characterized (see review
by Metcalf [1]).
Studies have succeeded in isolating cDNAs for
murine Multi-CSF [2, 3] and GM-CSF [4] and, using
each eDNA, biologically active recombinant CSF
has been produced in both mammalian and bacterial expression systems [5-10]. Purified, bacterially
synthesized GM-CSF has been shown at low concentrations (2-80 pg!ml) to be an effective stimulus
for granulocytic and macrophage colony formation,
at concentrations above 80 pg!ml to stimulate eosinophil colony formation, and at concentrations
above 640 pg!ml to stimulate megakaryocyte and
some pure and mixed erythroid colony formation
[ 10]. These properties were similar to those of native
GM-CSF at equivalent concentrations.
The present studies were undertaken to determine
whether the injection of purified, bacterially synthesized GM-CSF into normal adult mice was capable of stimulating detectable hemopoietic changes
corresponding to the known actions of this regulator
in vitro.
Materials and methods
Mice. Mice used were three-month-old males of the strains C57BL/
6fJ/WEHI, C3H/HeJ, and BALB/c/An/Bradley/WEHI maintained in this institute and were matched for body weight before
use. Mice had been reared under specific pathogen-free conditions
then conventionalized for five weeks before use.
Preparation and purification of bacterially synthesized murine
GM-CSF. A murine GM-CSF clone was isolated from EL-4 cells
using oligonucleotide probes based on the previously published
eDNA sequence for murine GM-CSF [4]. The production and
purification of recombinant murine GM-CSF synthesized by
Escherichia coli have been described previously [9, 10]. The purity to homogeneity of the recombinant GM-CSF was verified
by the following analytical procedures: refractionation using a
TSK HPLC column, peptide mapping, isoelectric focusing, amino acid sequence analysis, and quantitative amino acid analysis .
In no instance was evidence obtained of contaminating polypeptides.
Experimental Hematology vol. 15 (1987)
D. Metcalf et al.: Effects in vivo of GM-CSF
3
2
Examination of tissues. After performance of white cell counts,
mice were killed with ether. Following death, each mouse was
injected i.p. with 2 ml saline and, after gentle massage of the
abdominal cavity, the peritoneal wall was opened and as much
fluid as possible collected using a sterile pipette.
Bone marrow cells were collected from one complete femur
shaft and the spleen was removed aseptically. Portions of the
following tissues were taken for histological examination after
fixation in 10% formalin in saline-spleen, kidney, liver, mesenteric node, small bowel adjacent to the mesenteric node, lung,
heart, skin, and thymus. All sections were prepared in duplicate
and stained with hematoxylin-eosin or, for mast cell enumeration, with Alcian blue-safranin. Cytocentrifuge preparations were
made of peritoneal, bone marrow, and spleen cell suspensions
and stained with May-Griinwald-Giemsa.
Based on quantitative amino acid measurements, the recombinant GM-CSF (rGM-CSF) was diluted in 10% syngeneic mouse
serum/saline to produce a dilution containing 200 ng/0.2 ml.
During this dilution step, the material was processed through a
PD 10 column to remove residual acetonitrile. Serial dilutions of
this material were then made to contain 100, 50, 25, 12, and 6
ng/0.2 ml.
All batches of rGM -CSF prediluted in mouse serum/ saline and
the carrier serum/saline itself were assayed for endotoxin using
the Limulus amebocyte lysate assay both before and after courses
of in vivo injections and no endotoxin was detected (lower detection limit, 0.04 ng!ml). In previous control studies, mice injected with 0.2 ng endotoxin three times daily for six days failed
to develop any of the changes to be described, and a redescription
of these negative results [11] will not be repeated in the present
data although, in many of the experiments, control mice were
injected with 0.2 ng endotoxin, again with negative effects.
Bioassay of rGM-CSF levels. Levels of rGM-CSF in all preparations used for injection were assayed prior to and following
each course of injections using serial twofold dilutions of 0.1 ml
in 1 ml agar-medium cultures containing 75,000 C57BL bone
marrow cells [12]. Units (U) ofCSF per milliliter were calculated
from the linear portion of the dose-response curve, 50 U being
the concentration stimulating the formation of half-maximal
numbers of colonies. With the batches of purified recombinant
GM-CSF used, quantitative amino acid analysis indicated that
200 ng ofrGM-CSF assayed as 120,000 bone marrow units (specific activity 6 x 10 8 U/mg).
Bioassays on GM-CSF levels in the serum of injected mice
were performed using microwell cultures of 15 1'1 Dulbecco's
modified Eagle's medium containing 5 1'1 of serial serum dilutions
and 200 FDC-Pl cells [13]. Cell counts were performed on the
wells after 48 h of incubation and observed levels of GM-CSF
converted to bone marrow units per milliliter by use of standard
control preparations of GM-CSF of known activity.
Injection schedule. Mice were injected intraperitoneally (i.p.) with
0.2 ml of 10% mouse serum/saline containing 0-200 ng rGMCSF three times daily at 8:00A.M., 4:00 P.M. and 10:00 P.M. All
analyses were commenced at 9:00 A.M. on the morning following
the last (evening) injection.
White blood cell counts. Mice were anesthetized with ether and
orbital plexus blood collected using 50-f.Ll micropipettes. No mouse
was examined on more than one occasion to avoid misleading
cell counts from previously damaged and/or inflamed vessels.
White cell counts were performed using hemocytometers and
differential cell counts were performed on blood films stained
with May-Griinwald-Giemsa.
1000
Assay of progenitor cell levels in injected mice. Dispersed cell
suspensions were prepared from femoral marrow and a portion
of the spleen. Assays were performed in 1-ml agar cultures in 35mm petri dishes using 50,000 spleen cells or 25,000 marrow cells
per culture. The medium used was Dulbecco's modified Eagle's
medium with a final concentration of 20% fetal calf serum (FCS)
and 0.3% agar. Colony formation was stimulated by addition of
0.1 ml of a semipurified preparation of pokeweed-mitogen-stimulated spleen-conditioned medium containing 400 U GM-CSF
and 400 U Multi-CSF [14]. Cultures were incubated for seven
days in a fully humidified atmosphere of 10% C02 in air and
colony formation (clones >50 cells) scored at x 35 using a dissection microscope. The whole cultures were fixed with 2.5%
glutaraldehyde and then differential colony counts performed af-
e
g;_
~
l:l 100
:i::
'.:
2=>
0·5
Hours after injection
Fig. 1. Serum GM-CSF levels in BALB/c mice following a single intraperitoneal injection of 65 ng rGM-CSF. Serum GM-CSF
levels in uninjected BALB/c mice were undetectable in the assay
used ( < 5 U/ml). Mean values ± standard deviations.
Table 1. Peripheral blood white cell levels in BALB/c mice injected with rGM-CSF
Total cells/f.Ll
Injected with
Serum/saline 0.2 ml
rGM-CSF 200 ng
rGM-CSF 100 ng
Total cells/1'1
Neutrophils
9600
8770
10,160
8860
1540
1810
2840
2690
±
±
±
±
3990
2100
2050
2170
±
±
±
±
590
770
800
140
Lymphocytes
7500
6450
6740
5470
±
±
±
±
3260
1470
1730
1500
Eosinophils
60
90
110
60
±
±
±
±
80
80
100
60
Monocytes
500
420
400
480
±
±
±
±
300
210
170
270
BALB/c mice were injected three times daily for six days and then examined on day 7. Data from 12 mice per group ± standard
deviations. Neutrophil levels in mice injected with 200 ng rGM-CSF were significantly elevated above levels in either control group
(t = 4.5 and 3.4, respectively; p < 0.01). Levels in mice injected with 100 ng were not significantly different from those in mice injected
with serum/saline.
ter staining for acetylcholinesterase and counter-staining with
Luxol fast blue-hematoxylin.
Neutrophlls
Quantitative estimation of tissue content of megakaryocytes and
mast cells. Megakaryocytes and mast cells were counted in whole
spleen sections and then the organ content of these cells was
calculated from camera Iucida drawings as described previously
[II].
Phagocytosis assays. Pooled peritoneal cells from each treatment
group were washed twice in HEPES-buffered RPMI-1640 containing 2% FCS (RPMI-1640) and suspended at 10 6 cells/ml. Antibody-coated sheep erythrocytes (EA) were prepared by incubating
the cells with a pretitrated agglutinating amount of mouse IgG2b
anti-E (Sera-Lab MAS 013, Crawley Down, UK) for 40 min at
room temperature. The EA were then washed three times to
remove unbound antibody and suspended at 5 x I 0 7 cells/ml in
RPMI-FCS. Equal volumes (150 !'\) of peritoneal cells and EA
targets were added to Wasserman tubes and the cells were kept
at 4°C until the incubation (37°C, 10 min) was started after the
cells had been brought into contact by gentle centrifugation (500
g, 20 s). The assay was stopped by adding an excess of ice-cold
medium; extracellular erythrocytes were subjected to hypotonic
lysis and stained smears of the cells were prepared and scored as
previously described [11, 15].
Results
Serum levels of rGM-CSF in injected mice
BALB/c mice were given a single i.p. injection of
65 ng rGM-CSF and then the serum was collected
at intervals up to 6 h and assayed in microwell cultures ofFDC-P1 cells to determine GM-CSF levels.
Serum GM-CSF levels in uninjected BALB/c mice
were undetectable ( < 5 U/ml). As shown in Figure
1, levels of GM-CSF in the serum rose to a peak
value of 500 U/ml 30 min following injection, then
fell logarithmically with a half-life of approximately
35 min to 10 U/ml by 3 h after injection. Based on
these observations, the range of doses of rGM -CSF
chosen for injection was from 6 ng to 200 ng.
Peripheral blood changes
White cell counts were performed on BALB/c mice
after six days of injection of 200 or 100 ng rGMCSF. As shown in Table 1, the only significant change
observed was a slightly less than twofold elevation
in neutrophil levels. No immature granulocytes or
nucleated red cells were observed in the blood.
Peritoneal cell changes
Groups of BALB/c mice were injected with 6-200
ng. rGM-CSF three times daily for six days and the
m1~e examined on day 7. The data on the peritoneal. cell populations in mice from three such experiments have been pooled in Figure 2. Clear
30
'§
0
;2 20
10
GM- CSF per injection ng
Fig. 2. Peritoneal cell population changes in BALB/c mice injected intraperitoneally for six days with varying doses ofrGMCSF: U, uninjected mice; and S, mice injected with 0.2 ml mouse
serum/saline. Each point is mean value from 12 mice± standard
deviations. Neutrophil levels in mice injected with 50, 100, and
200 ng rGM-CSF were significantly higher than in serum/salineinjected mice (t = 3.2, 3.9, 2.8, respectively; p < 0.01). Macrophage levels in mice injected with 6, 12, 25, 50, 100, and 200
ng rGM-CSF were significantly higher than in serum/saline-injected mice (t = 5.1, 9.0, 5.5, 6.5, 7.9, and 8.0, respectively; p <
0.01). Eosinophil levels in mice injected with 6, 12, 25, 50, 100,
and 200 ng rGM-CSF were significantly higher than in serum/
saline-injected mice (t = 5.4, 4.7, 3.8, 5.6, 3.9, and 4.5, respectively; p < 0.01).
dose-related rises were observed in peritoneal macrophages, eosinophils, and neutrophils, with no significant changes in lymphocyte levels. Rises in macrophage numbers were significant even with the
lowest dose used (6 ng/injection) and, with the highest dose, macrophage levels reached 50 x 106 cells
in some mice. The data suggested that plateau responses had not been achieved even with the highest
dose used. The neutrophil and eosinophil rises
reached levels 100-fold higher than in uninjected
control mice and 10- to 20-fold higher than in mice
injected with serum/saline and involved exclusively
postmitotic mature cells in both lineages (Fig. 3).
The peritoneal macrophages from mice injected with
rG M -CSF were larger and more basophilic than resident macrophages and many exhibited cytoplasmic
vacuolation. Significant mitotic activity was observed in peritoneal macrophages from mice injected with rGM-CSF, reaching mean levels of 7.6/
103 cells, although no distinct relationship was observed between the dose of rGM-CSF injected and
the frequency of mitotic figures (Table 2).
Experimental Hematology vol. 15 (1987)
D. Metcalf et al.: Effects in vivo of GM-CSF
5
4
Table 2. Elevated mitotic activity in peritoneal macrophages
from BALB/c mice injected with rGM-CSF
Injected with
Observations on groups ofC57BL and C3H/HeJ
mice injected with the same dose range ofrGM-CSF
gave similar results to those described for BALB/c
mice. In both strains, significant rises in peritoneal
macrophages and eosinophils were observed with
32
Mitoses/ 10 3 macro phages
0.2 ml serum/saline
0±0
0.2 ± 0.7
rGM-CSF
6 ng
12 ng
25 ng
50 ng
100 ng
200 ng
6.5
5.0
4.0
4.2
7.6
5.1
±
±
±
±
±
±
28
rGM-CSF
5.4
3.9
4.0
4.5
7.5
5.8
Twelve mice per group were injected three times daily for six
days and examined on day 7. Mean frequency of mitotic figures
per 10 3 macro phages ± standard deviations (300-600 cells scored
in each preparation). Frequency of mitoses in cells from mice
injected with 6, 12, 25, 50, and 100 ng rGM-CSF were significantly higher than in cells from control mice injected with serum/
saline (t = 3.8, 4.0, 3.0, 2.9, and 3.3, respectively; p < 0.01).
Fig. 3. Changes induced in BALB/c mice following the intraperitoneal injection for six days of 200 ng GM-CSF. (A) Peritoneal cells from mouse injected with rGM-CSF showing increased macrophages (one in mitosis), neutrophils, and eosinophils
(arrows). (B) Peritoneal cells from control mouse injected with
serum/saline. (C) Liver from mouse injected with rGM-CSF
showing increased infiltration of granulocytes and monocytes.
(D) Liver from control mouse. (E) Lung from mouse injected
with rGM-CSF showing increased cellularity of alveolar sac walls,
including neutrophils. (F) Lung from control mouse. Cytocentrifuge preparations stained with May-Griinwald-Giemsa, sections with hematoxylin-eosin.
Peritoneal Macrophages
36
the lowest dose used (6 ng) and with 200 ng the
mean levels of cells were as follows. C57BL: macrophages 43.2 ± 10.2 x 106 , neutrophils 0.4 ±
0.3 x 106 , and eosinophils 4.8 ± 1.7 x 106 (control
C57BL mice injected with serum/saline: macrophages 3.1 ± 1.0 x 106 , neutrophils 0.07 ± 0.08 x
106 , and eosinophils 0.01 ± 0.03 x 106 cells). C3H/
HeJ: macrophages 16.4 ± 2.4 x 106 , neutrophils
0.3 ± 0.3 x 106 , and eosinophils 1.3 ± 0.3 x 106
cells (control C3H/HeJ mice injected with serum/
saline: macrophages 2.0 ± 1.1 x 106 , neutrophils
0.05 ± 0.06 x 106 , and eosinophils 0.01 ± 0.02 x
106 cells). In neither strain were significant changes
observed in levels of peritoneal lymphocytes.
Sequential studies on C57BL mice injected with
200 ng rGM-CSF showed that, by 3 h after a single
injection, no significant changes had occurred in
peritoneal cell levels, indicating that no rapid immigration of cells to the peritoneal cavity was induced by rGM-CSF. In mice injected three times
daily, minor elevations of peritoneal macrophages
were observed at 24 h and levels rose progressively
thereafter (Fig. 4). A similar pattern was observed
for neutrophils and eosinophils.
Phagocytic activity of peritoneal macrophages
Assays were performed on the ability of peritoneal
macrophages to phagocytose antibody-coated sheep
erythrocytes using cells harvested from BALB/c,
C57BL, and C3H/HeJ mice injected for six days
with varying doses ofrGM-CSF. Typical results from
one such experiment with cells of each strain are
shown in Figure 5. The absolute levels of phagocytic
activity in each of the three experiments are not
·""
rGM·CSF per 1nJect1on ng
3
4
Days of Injection
4: .Progressive rise in total peritoneal macrophages in C57BL
m1ce Injected three times daily with 200 ng rGM-CSF or 0.2 ml
mouse serum/saline. Each point represents an individual mouse.
Fi~.
directly comparable because of the use of different
batches of antibody. Clear evidence was obtained
with cells from each strain that rGM-CSF induced
dose-related rises both in the percentage of macrophages with phagocytosed erythrocytes (up to
eightfold) and in the average number of erythrocytes
per p~ag~cytically active cell (up to sevenfold).
Combmatwn of these data with the absolute rises
in total macrophages induced by rGM-CSF indicated that the overall level of phagocytic capacity
m the total peritoneal cavity population had been
increased in excess of 100-fold.
Fig. 5. Phagocytic activity for antibody-coated erythrocytes of
pooled _veri.t~neal macrophages from BALBIc, C5 7BL, and C3H/
HeJ mice Injected three times daily for six days with varying
doses of rGM-CSF: U, uninjected; and S, injected with 0.2 ml
mouse serum:s~l.ine. Note progressive rise in percentage ofmacroph~ges exhibitmg phagocytic activity (e----e) and the rGMCS~-mduce~ rise in the average number of erythrocytes in phagocytically active macrophages (0--0). Each point is mean value
from duplicate preparations. The absolute values for each mouse
str~in are not directly comparable because of the use of differing
antibody preparations.
18
16
14
~
i'"
12
~
~ 10
d
E
f
8
~
6
~
H
§
z
Liver changes
In BALB/c mice injected for six days with varying
doses ofrGM-CSF, a dose-related rise was observed
m th~ number ofnonparenchymal cells in the liver.
The mfiltrating cells were mainly neutrophils and
macrophages with some eosinophils and occasional
megakaryocytes (Fig. 3). The infiltrating cells were
usually dispersed between the parenchymal cords
but were also present as focal aggregates not nee~
essari.ly adjacent to portal vessels. No mitoses were
seen m these cells. Counts on the number of nonparenchymal cells were performed avoiding focal
a.ggre~ates but, even with this deliberate underestlm~tl?n, a rise of at least 50% was observed in mice
receiVmg the higher doses ofrGM-CSF (Fig. 6).
u s
6 12 25 50 100200
rGM-CSF per injection ng
Fig. 6. Dose-related rise in nonparenchymal cells in the liver
of B~LB/c mice injected three times daily for six days with
:arymg d~ses of rGM-CSF: U, uninjected mice; and S, mice
InJected With 0.2 ml mouse serum/saline. Each point represents
mean cell counts per six high-power fields from each of 12 mice +
standard deviations. Numbers in mice injected with 100 and 200
~g rGM~CSF were significantly higher than in control mice inJected With serum/saline (t = 4.7 and 5.0, respectively;p < 0.01).
It proved impossible to quantify this increased cell~lar infiltration because of the irregular compres~lOn of the alveolar sacs in the sections, but the
I~creased numbers of infiltrating neutrophils per
high-power field approximated two- to fivefold.
Lung changes
In BALB/c mice injected with the higher doses of
rG?vf-CSF, it was common for the alveolar sac walls
to b em
· filtrated by increased numbers of cells the
most readily identifiable of which were neutro~hils.
Spleen changes
Injection of BALB/c mice for six days with rGMCS~ resulted in a moderate dose-related rise in spleen
weight approaching 50% (Table 4). Because of the
Experimental Hematology vol. 15 (1987)
6
Table 5.
Table 3.
7
D. Metcalf et al.: Effects in vivo of GM-CSF
Effects ofrGM-CSF on bone marrow cellularity in BALB/c mice
Effect of rGM-CSF on cell populations in the spleen of BALB/c mice
Total cells per femur x
Percent cells
Group
Uninjected
Serum/saline
rGM-CSF
Spleen
weight (mg)
Blasts
111 ± 18
102 ± 13
0.5 ± 0.6
0.5 ± 0.5
MetamyeloPromyelocytes myelo- cytes neutrophils
cytes
0.2 ± 0.4
0.4 ± 0.8
6.1 ± 3.6
6.1 ± 2.2
Lymphocytes
Monocytes
Eosinophils
Nucleated
red cells
76.4 ± 4.4
78.9 ± 4.8
3.5 ± 1.9
2.5 ± 1.4
0.4 ± 0.7
0.4 ± 0.7
12.9 ± 4.7
11.2 ± 4.2
7.9 ±
9.3 ±
12.0 ±
17.1 ±
17.3 ±
16.7 ±
3.3
3.6
4.6
6.0
5.9
7.8
± 2.1
0.5
0.3
0.4
0.1
0.2
0.3
±
±
±
±
±
±
0.5
0.5
0.7
0.3
0.4
0.5
2.3
4.3
6.2
7.9
7.5
5.1
85.1 ± 4.0
0.8 ± 0.1
0.4 ± 0.5
2.0 ± 2.2
± 2.6
82.8 ± 6.7
6 ng
0.4 ± 0.7
3.1 ± 1.8
0.5 ± 0.8
12 ng
± 2.9
79.3 ± 7.1
0.2
±
0.4
2.4
±
1.4
25 ng
1.1 ± 0.9
± 2.3
72.7
±
10.2
2.9 ± 4.0
0 ·6 +
0.6 ± 0. 9
- 0 ·9
5o ng
± 4.0
63.1
±
7.2
1.3±1.4
0.5±0.7
1!.7±3.1
± 2.8
100 ng
8
52
63.2 ± 9.3
200 ng
1.2 ± 0.8
1.0 ± 0.6
9. ± ·
.
.
. . ed three times per day for six days and examin~d _on day
Percent
Mean data from 12 BALB/c mlce per group. Mlce were mject
. 'fi ntly higher than in control mice mJected wlth serum/
monocytes for mice injected with 50, 100, and 200 ng rGM-CSlF
Slytg~~ ~:mice injected with 100 and 200 ng rGM-CSF were
d 58
spectively- P < 0 01) Percent ymp oc
01)
8
saline (t = 4.4, 2. , an
· • re
. . .'
: ·
·
1 r e (t = 6 4 and 5.2, respectively; P < 0.
significantly lower than in control m1ce mjected Wlth serum sa m
·
101 ± 14
109 ± 26
106 ± 22
146 ± 29
128 +40
153 ± 30
?.
we:
Table 4.
Mast cells per spleen
Group
Megakaryocytes
per spleen
Large
111 ± 18
102 ± 13
89 ± 49
89 ± 40
13 ± 10
14 ± 11
Uninjected
Serum/saline
Small
10 ± 7
14 ± 9
Nonerythroid progenitor cells per
5 x 104 cells
!.7±1.5
1.0 ± 1.0
rGM-CSF per injection
1.5 ± 0.7
3 ± 1
3 ± 1
± 5
2.7 ± 2.9
12
±
7
12
±
7
-± 66
1.2 ± 0.3
8 ± 8
5 ± 3
-+ 27
5.2 ± 4.5
13
±
10
11
±
10
-+ 50
5.0 ± 2.8
9±6
9 ± 5
± 49
4.3 ± 2.3
126 ± 40
+ 71
15 ± 15
9±9
153 ± 30
241 .
. .
d three times per day for six days with the stated amount of rGM-CSF
Mean data from 12 BALB/c mice per group. Mlce were mJecte
b
1 lated from cell counts on spleen sections of known area
and examined on day 7. Relative megakaryocyte and mast cell num ~rs ca cu . nificantly higher than for serum/saline-injected mice
1
. hts for 50 ng and 200 ng mlce were Slg
. h
h f,
m/
as described previously [11 l . SPeen welg
f, 100
and 200 ng mice were significantly hlg er t an or seru
(t = 4.7 and 4.2, respectively; p < 0.01). Meg~aryocyte numbers or
ng
saline-injected mice (t = 4.5 and 6.5, respectively; P < 0.01).
6 ng
12
ng
25
ng
0
5 ng
100 ng
200 ng
101
109
106
146
±
±
±
±
14
26
22
29
70
137
92
119
179
need to examine spleen sections from these mice,
total spleen cell counts were not perfor~ed. _Ho_wever parallel experiments showed that, m m1ce mject~d with rGM-CSF, spleen weight increases were
paralleled by corresponding rises in total cell counts.
Differential cell counts revealed a consistent twofold
rise in the percentage of monocytes, and small nonsignificant rises in immature granulo~y~es, blast cells,
and nucleated erythroid cells. Surpnsmgly, no consistent elevations were observed in the percentag~s
of mature neutrophils or eosinophils (Table 3). Th1s
same response pattern was seen in th.e spleen of
C57BL and C3H/HeJ mice where agam the most
consistent change was a two- to fourfold rise in percent monocytes.
Monocytes
Eo sinophils
11.0 ± 1.9
8.4 ± 1.3
1.7 ± 0.7
1.9 ± 0.9
0.7 ± 0.6
l.l ± 0.7
5.7 ± 1.8
7.4 ± 3.7
29.8 ± 9.0
26.2 ± 14.1
1.9
1.4
1.5
2.2
1.6
1.7
0.7
0.6
0.6
0.7
0.6
0.9
4.6
4.5
4.0
4.1
2.2
1.9
14.5
10.6
13.4
12.8
12.1
9.5
Uninjected
Serum/saline
29.0 ± 4.6
31.7 ± 8.0
0.7 ± 0.3
0.5 ± 0.4
1.6 ± 0.4
1.7 ± 0.6
7.6 ± 2.5
10.7 ± 2.3
rGM-CSF
6 ng
12 ng
25 ng
50 ng
100 ng
200 ng
25.0 ± 11.3
24.0 ± 4.6
19.7 ± 2.9
25.3 ± 2.3
19.3 ± 2.1
17.0±7.0
0.3
0.6
0.4
0.4
0.5
0.3
1.2
1.2
1.5
1.4
1.0
1.2
8.1
11.0
7.6
12.3
9.9
9.4
0.2
0.3
0.2
0.3
0.2
0.1
±
±
±
±
±
±
0.6
0.8
0.5
0.6
0.4
0.4
±
±
±
±
±
±
In sections of the spleen from mice injected :-'ith
rGM-CSF, the tissue retained the normal architectural pattern oflymphoid follicles and red pulp, but
counts revealed a dose-related rise in the number
of megakaryocytes, reaching a two- to threefold elevation in mice injected with 200 ng rGM~CS~ <!able 4). In sharp contrast to the IOO~fol~ :1se m 1~­
mature mast cells in the spleen ofm1ce l_nJected w1th
rMulti-CSF [11], no rise was observed m t_nature or
immature mast cells in mice injected w1th rGMCSF (Table 4).
.
.
Assays for nonerythroid progemtor cells 1~ t~e
spleen revealed a moderate (u~ to fourfold) nse m
BALB/c mice receiving the h1gher doses_ of rGMCSF (Table 4). No change was observed m the rei-
2.6
0.9
1.4
2.4
1.3
4.1
8.2
4.7
4.1
4.2
3.5
1.7
±
±
±
±
±
±
4.2
1.6
1.1
1.2
1.6
0.6
±
±
±
±
±
±
l.l
0.7
0.5
0.8
0.5
0.4
±
±
±
±
±
±
0.4
0.5
0.4
0.3
0.4
0.6
±
±
±
±
±
±
1.8
2.1
1.4
l.l
0.9
0.9
±
±
±
±
±
±
6.4
1.6
8.7
4.7
8.3
2.7
BALB/c mice were injected with stated material three times daily for six days and examined on day 7. Mean data from 12 mice per
group± standard deviations. Lymphocyte levels in mice injected with 12, 25, 50, 100, and 200 ng rGM-CSF were significantly lower
than in control mice injected with serum/saline (t = 6.0, 8.9, 8.2, 8.3, and 16.6, respectively; p < 0.01). Nucleated erythroid cell levels
in mice injected with 25, 50, 100, and 200 ng rGM-CSF were significantly lower than in control mice injected with serum/saline (t =
3.0, 2.9, 4.8, and 5.0, respectively, p < 0.01).
ative frequencies of granulocyte, macrophage, or eosinophil colony-forming cells.
.
d mast cells in BALB/c mice
Effects ofrGM-CSF on spleen we1ght, megakaryocytes, an
Spleen weight
(mg)
Lymphocytes
Blasts
±
±
±
±
±
±
Total
nonerythroid
progenitor
Nucleated
cells per
red cells femur x IQ- 3
Promyelo- Metamyelocytes my- cytes polyelocytes
morphs
Total cells
Group
IQ-6
Bone marrow changes
In BALB/c mice injected for six days with rGMCSF, a dose-related fall was observed in total femur
cell counts. Levels of blast cells, immature and mature granulocytes, monocytes, and eosinophils remained constant and the fall in cellularity was due
to a fivefold reduction in marrow lymphocytes and
fourfold reduction in nucleated erythroid cells (Table 5). Absolute levels of nonerythroid progenitor
cells also showed a progressive fall with increasing
doses ofrGM-CSF reaching levels 64% below those
in control mice (Table 5). Differential colony counts
revealed no change in the relative frequency of granulocytic, macrophage, or eosinophil colonies. In
C57BL mice, absolute progenitor cell numbers were
reduced 37% and in C3H/HeJ mice 66% by the
highest doses ofrGM-CSF.
Changes in other organs
In BALB/c mice injected for six days with rGMCSF, no infiltration of hemopoietic cells was observed in the mesenteric lymph node, heart, kidney,
gut, or skin.
Dl:scussion
In vitro studies using bacterially-synthesized GMCSF have indicated that this material has the same
specific activity per milligram of protein as native
GM-CSF. In addition to the colony-stimulating activity summarized in the introduction, both forms
of GM-CSF also have a weak ability to generate
committed progenitor cells from multipotential cells
[14, 16].
Although the effects of injected rGM-CSF cannot
be proved to be direct effects and might in part have
been due to interactions with other factors or to
indirect effects, the observed responses were in fact
in agreement with expectations from the known in
vitro direct actions ofGM-CSF and were similar in
the three strains tested- BALBIc, C5 7BL, and C3H/
HeJ. The responses observed are summarized in
Table 6 and have some similarity with those previously observed in mice injected with comparable
doses of rMulti-CSF [11]. However, major differences were that immature mast cells in the spleen
were increased 100-fold by rMulti-CSF, but unaffected by rGM-CSF, and that nonerythroid progenitor cell levels in Multi-CSF-injected mice remained
normal in the marrow and were elevated 10- to 20fold in the spleen whereas, in mice injected with
rGM-CSF, progenitor cell levels rose less markedly
in the spleen and were significantly depleted in the
marrow.
Assays for endotoxin failed to detect endotoxin
in any of the preparations injected (lower detection
limit, 0.04 ng/ml) and the injection of endotoxin in
even higher doses (1 ng/ml) failed to affect the levels
of any of the cells listed in Table 6 [11]. Furthermore, endotoxin-hyporesponsive C3H/HeJ mice
showed similar changes after the injection of rGMCSF to those in other strains. The distinct differences in responses to injected rMulti-CSF and rGM-
Experimental Hematology vol. 15 (1987)
D. Metcalf et al.: Effects in vivo of GM-CSF
9
8
Table 6. Summary of changes induced by the injection of rGMCSF for 6 days•
Parameter
Peripheral blood
Neutrophils
Peritoneal cavity
Macrophages
Neutrophils
Eosinophils
Macrophage mitotic
activity
Macrophage phagocytic
activity
Liver
Hemopoietic cells
Lung
Neutrophils
Spleen
Weight
Monocytes
Megakaryocytes
Nonerythroid progenitor
cells
Bone marrow
Total cells
Nucleated erythroid cells
Lymphocytes
Nonerythroid progenitor
cells
Rises
Falls
2-fold
15-fold
l 0- to l 00-fold
l 0- to l 00-fold
40-fold
100-fold
50%
2- to 5-fold
50%
2- to 4-fold
2- to 4-fold
2- to 5-fold
40%
3-fold
5-fold
37%-66%
• Relative to levels in control mice injected with serum/saline
alone.
CSF (both produced by the same strain of E. coli)
in themselves constitute strong evidence that the
responses observed were not ascribable to undetected contaminants of bacterial origin.
A single i.p. injection of65 ng rGM-CSF achieved
peak serum GM-CSF levels of 500 U/ml 30 min
after injection which fell by 3 h to levels of 10 U /ml,
a concentration in vitro that would only have minimal effects on hemopoiesis. Even with the highest
doses used (200 ng), the interrupted injection schedule used would only have achieved a serum concentration of GM-CSF considered from in vitro
studies to be significant (greater than 50 U/ml) for
periods of up to 3 h, following which a period of 57 h would then elapse with no significant elevation
of CSF levels. The transient nature of these elevations probably reduced the magnitude of the changes
observed.
Quantitatively, the most striking response to injected rGM-CSF involved cells of the peritoneal
cavity, the site ofCSF injection. The elevated numbers of neutrophils and eosinophils exclusively in-
valved cells that were postmitotic and these must
have slowly accumulated in the cavity from the circulation. Human GM-CSF has been shown to inhibit neutrophil migration [17] and GM-CSF may
possess previously unsuspected direct or indirect
chemotactic effects. In the case of the peritoneal
macrophages, clear mitotic activity was observed
(mitotic index up to 0.7%). Depending on the duration of the mitotic event, this frequency of mitoses
could represent local proliferative activity sufficient
to account for at least half of the rise in macrophages
(if mitosis lasted 30 min), but would account for a
smaller proportion of the observed rise if mitosis
lasted longer.
The peritoneal macrophages in mice injected with
rGM -CSF exhibited clearly increased phagocytic activity for antibody-coated sheep erythrocytes. This
observation was similar to that made previously in
mice injected with rMulti-CSF [11] and supports in
vitro evidence that the CSFs are able to stimulate
the functional activation of mature granulocytes and
macrophages [1].
There was a moderate increase in the relative and
absolute numbers of granulocyte-macrophage progenitors in the spleen, but this was more than balanced by a fall in the absolute number of progenitor
cells in the marrow of approximately 60%. Thus the
increased numbers of granulocytes and macrophages in mice injected with rGM-CSF appear to
have been achieved at the expense of some depletion
of the total population of granulocyte-macrophage
progenitor cells. It will require studies oflonger duration to determine whether this depletion process
would continue if injections ofrGM-CSF were continued for longer periods or whether stem cell activation would be triggered with some restoration
of progenitor cell levels. To clarify this situation,
observations are also needed on stem and multipotential cell levels in mice injected with rGM-CSF.
The spleen is the characteristic organ in adult mice
exhibiting increased hemopoiesis in response to
stress or stimulation. One curious aspect of the response pattern to injected rGM-CSF was the small
change in granulocytic or monocytic cells in the
spleen in contrast to the marked rises in these populations in the peritoneal cavity, liver, and lungs.
Even in the marrow, absolute numbers of granulocytes remained constant. Thus the stimulation of
granulocytic and monocytic populations by rGMCSF, at least at the time points sampled, did not
increase these populations in the organs generating
these cells (the marrow and spleen) but, instead,
resulted in clear increases of these cells in peripheral
tissues such as the peritoneal cavity, liver, and lung.
In the context of the possible use of rGM-CSF in
an animal suffering from a refractory infection, this
pattern of the response is quite advantageous in increasing the numbers of protective granulocytes and
monocytes in locations that might be sites of a widespread infection.
With the lower doses of rGM-CSF used, the expected serum CSF levels would at maximum still
be in the linear portion of the in vitro dose-response
curve. The ability of these relatively small doses of
rGM-CSF, even when separated by relatively long
intervals, to induce detectable changes suggests that
the responsiveness to rGM-CSF of at least some in
vivo parameters may not be dissimilar from that
exhibited by hemopoietic populations in vitro. The
range of serum concentrations achieved by the injection of rGM-CSF are within those actually observed in the serum during responses to natural viral
and bacterial infections [12].
These present responses induced in normal mice
injected rGM-CSF strongly support the conclusion reached from extensive earlier indirect observations [12] that GM-CSF is likely to function in
vivo as a genuine regulator of granulocyte-macrophage populations and possibly some related hemopoietic populations. In the clinical context the
administration ofrGM-CSF can be expected t~ result in useful stimulation of hemopoietic populations in patients with subnormal hemopoiesis, e.g.,
following cytotoxic therapy, and in patients with
refractory infections.
Acknowledgments
The authors are indebted to the careful technical assistance of
Mrs. C. Quilici and Miss Y. Wiluszynski. This work was supported by the Carden Fellowship Fund of the Anti-Cancer Council of Victoria, The National Health and Medical Research Council, Canberra, and the National Institutes of Health, Bethesda,
grants CA-22556 and 25972.
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